Monoclonal antibodies are powerful therapeutic agents in the treatment of cancer since they selectively target antigens which are differentially expressed on cancer cells. Targeting of the TRAIL (TNF related apoptosis inducing ligand) death receptors on cancer cells with agonistic monoclonal antibodies represents a new generation of monoclonal antibody therapy, as they are able to directly induce apoptosis of targeted cells.
Upon binding of TRAIL, death receptors of the TNFR-SF family such as DR4 and DR5 become trimerized. The trimerization induces the extrinsic apoptotic pathway and a complex cascade of events including Caspase activation, which finally result in the killing of the target cells. Apoptosis induction is further enhanced if hyperclustering of DR5 (i.e. the clustering of multiple trimers) takes place. Although death receptors are widely expressed on a variety of cell types, induction of apoptosis via the extrinsic pathway is restricted to tumor cells. Since agonistic DR4 or DR5 binding antibodies are able to cross-link death receptors and hence induce apoptosis, these receptors are interesting targets in cancer therapy. At least eight death receptor targeting molecules entered clinical development and have been assessed in clinical trials for possible treatment of different indications such as advanced solid tumors like colorectal or lung cancers. In addition there have been attempts to treat other indications such as lymphoma and/or multiple myeloma.
Drozitumab, a fully human DR5 agonistic antibody described in US2007/0031414 A1 and WO2006/083971, shows some in vitro apoptotic activity in the absence of cross-linking at high concentrations. However, in vivo data revealed a different mode of action: In FcγR mutant mice (or when antibody variants were used in which FcγR binding was inhibited) Drozitumab was inactive indicating that the in vivo activity of this molecule is mainly dependent on FcγR mediated cross-linking. This molecule was tested up to clinical phase II, seemed to be save (no MTD up to 20 mg/kg was reached) but did not demonstrate any significant efficacy.
Conatumumab (described in EP1922337A), is another fully human DR5 agonistic antibody. The activity of Conatumumab is strictly dependent on cross-linking via Fc receptors. In contrast to Drozitumab this antibody is non-ligand blocking. Also this molecule only showed very limited efficacy in clinical trials.
LBY-135, a chimeric DR5 antibody, exhibits similar characteristics as Conatumumab with respect to cross-linking dependent activity and non-ligand blocking property and did not demonstrate any significant efficacy in monotherapy. In addition, LBY-135 showed signs of immunogenicity in part of the enrolled patients of a phase I trial.
Dulanermin, a recombinantly produced natural ligand of DR4 and DR5 (TRAIL), only showed limited objective responses in clinical trials. The use of the natural ligand has somehow disadvantageous: TRAIL targets multiple receptors including both the death receptors and decoy receptors and, therefore, selectivity is a concern. In addition, TRAIL has a much shorter blood half-life compared with monoclonal anti-DR antibodies, a factor which affects dose and schedule parameters. The very short blood half-life of TRAIL requires large and frequent doses compared with monoclonal anti-DR antibodies. In addition recombinant TRAIL is very difficult and tedious to produce.
The development of all three DR5 agonistic antibodies and the ligand described above was discontinued.
Two additional fully human antibodies, Mapatumumab (anti DR4) and Lexatumumab (anti DR5) are still in development although also these molecules did not exhibit promising efficacy in monotherapy.
Tigatuzumab is a humanized DR5 agonistic antibody which is described as being active in vitro (already at low concentrations) in the absence of secondary cross-linking which of course bears the risk of systemic toxicity issues. However, as all the other described agonistic DR5 antibodies, also this molecule has not demonstrated convincing efficacy in Ph I/Ph II studies so far and the maximally tolerated dose MTD only was demonstrated up to 8 mg/kg.
A different approach to induce apoptosis by targeting a death receptor is pursued with the molecule TAS266, a tetrameric DR5 binding nanobody (WO2011098520A1). Due to the tetravalent configuration of DR5 binding moieties, it is thought that DR5 cross-linking is increased compared to standard bivalent antibodies, which may result in increased activity. However, due to their small size, these molecules have the disadvantage of a rather short half-life (compared to antibodies). In addition there is an increased risk of systemic toxicity since this tetrameric molecule is not targeted to the tumor.
Combining the DR5 antibody Drozitumab with a tumor antigen binding moiety or an antigen present in the stroma surrounding the tumor in a bispecific antibody platform has been described by the inventors of the present application as a new approach to achieve two effects: firstly the DR5 binding antibody can be targeted to the tumor site which could avoid potential systemic toxicity issues (especially when using a DR5 antibody exhibiting cross-linking independent activity). Secondly, this tumor or tumor stroma targeting moiety then also serves as the cross-linking unit to induce DR5 hyperclustering and subsequently tumor site specific apoptosis. The basic concept has been demonstrated using Drozitumab_scFv fusion molecules targeting different tumor types (see WO 2011/039126).
Of particular interest therein were bispecific antibodies binding DR5 and Human Fibroblast Activation Protein (FAP; GenBank Accession Number AAC51668). Human FAP was originally identified in cultured fibroblasts using the monoclonal antibody (mAb) F19 (described in WO 93/05804, ATCC Number HB 8269). Homologues of the protein were found in several species, including mice (Niedermeyer et al., Int J Cancer 71, 383-389 (1997), Niedermeyer et al., Eur J Biochem 254, 650-654 (1998); GenBank Accession Number AAH19190). FAP has a unique tissue distribution: its expression was found to be highly upregulated on reactive stromal fibroblasts of more than 90% of all primary and metastatic epithelial tumors, including lung, colorectal, bladder, ovarian and breast carcinomas, while it is generally absent from normal adult tissues (Rettig et al., Proc Natl Acad Sci USA 85, 3110-3114 (1988); Garin-Chesa et al., Proc Natl Acad Sci USA 87, 7235-7239 (1990)). Subsequent reports showed that FAP is not only expressed in stromal fibroblasts but also in some types of malignant cells of epithelial origin, and that FAP expression directly correlates with the malignant phenotype (Jin et al., Anticancer Res 23, 3195-3198 (2003)). Surprisingly the inventors found that a bispecific antibody targeting FAP in the stroma and DR5 on the tumor cell actually induces apoptosis despite the targets being situated on different cells.
Upon further investigation the inventors of the present application found that the scFv containing bispecific molecules described in WO2011/039126 all have some intrinsic issues with respect to productivity, stability and aggregate formation leading to suboptimal, non-specific activity.
In the present application, novel bispecific antibodies targeting FAP and DR5 are provided. The inventors of the present application developed novel DR5 binding moieties that are only active after crosslinking. Hence induction of tumor cell apoptosis is dependent on DR5 hypercrosslinking via FAP and is independent on Fc/FcR interactions. Therefore in addition to bispecific antibodies specific for FAP and DR5, also novel antibodies binding to DR5 are provided therein.
In contrast, the activity of conventional DR5 targeting molecules as described above is dependent on Fc Receptor (FcR) mediated hyperclustering, and is influenced by the immune infiltration and activation status in the tumor (Li and Ravetch, PNAS 2012; Wilson, Cancer Cell 2011; WO2011098520A1). The Fc/FcR interactions can be impaired by physiological human IgG levels. Thus the activity of conventional DR5 targeting molecules is often limited to a few infiltrating cells (Moessner, Blood 2010). By using a bispecific antibody targeting both DR5 and FAP, the percentage of sensitive tumor cells can be significantly increased by hypercrosslinking via FAP and the risk of an intrinsic resistance to DR5 agonists is decreased. The novel DR5 binding moieties are only active after crosslinking with FAP, which could result in an improved safety and toxicology profile compared to the DR5 binders Apomab and Tigatuzumab. The DR5 agonists that have been tested so far were safe in the clinic, however, these clinical programs have been impeded by a low efficacy of the DR5 targeting molecules.
In addition, the preferred novel DR5 binding moieties bind to a different epitope than Drozitumab.
Importantly the novel DR5 binding moieties can be employed in many bispecific DR5-FAP targeting antibody formats, including both novel and established bispecific formats. In contrast, only C-terminal fusions of a FAP binding moiety are possible with Drozitumab-based bispecific DR5-FAP targeting antibody formats, as any N-terminal fusion to Drozitumab results in inactive molecules. The provision of the new DR5 binding moieties thus significantly expands the possibilities of employing the DR5 targeting moiety in various bispecific DR5-FAP targeting antibody formats. This is particularly important as some bispecific antibody formats have superior characteristics in terms of producibility and activity.
Provided therein are novel bispecific antibodies comprising novel DR5 binding moieties and a affinity matured FAP binding moiety.
The bispecific antibodies of the present invention are provided in a bispecific antibody format, wherein one or more crossover-Fab fragments are fused to an IgG molecule. Crossover Fab fragments are Fab fragments wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Bispecific antibody formats comprising crossover Fab fragments have been described, for example, in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010/136172, WO2010/145792 and WO2013/026831.
So far, only DR5-FAP bispecific antibodies in a scFv containing format have been described (WO2011/039126). In addition to the advantageous properties of the novel DR5 binders disclosed therein, use of a crossover-Fab based bispecific format results in improved yield and less aggregates and side-products during production. In addition, scFv- and scFab-based DR5-FAP targeting bispecific antibodies demonstrated the tendency to form aggregates and therefore bear a higher risk to non-specifically crosslink DR5 even in absence of FAP. Hence these formats have the disadvantage of potentially inducing apoptosis in non-target cells (i.e. healthy cells).
The DR5 and FAP binding moieties of the novel bispecific antibodies provided herein exhibit superior in vivo efficacy compared to conventional DR5 antibodies. The preferred bispecific antibodies of the present invention bind with a high affinity to FAP on the tumor stroma and with a lower affinity to DR5 on the tumor cell. Moreover, the DR5 and FAP targeting bispecific antibodies provided herein do not crossreact with closely related proteins DR4, DcR1, DcR2 (closely related to DR5) and DPPIV (closely related to FAP). In addition it is now for the first time possible to provide a DR5-FAP bispecific antibody in various bispecific formats with no limitation as to the number of valencies of each binding specificity.
To summarize, the novel bispecific DR5-FAP antibodies provided therein are highly specific and potent: They selectively induce apoptosis by DR5 hyperclustering in tumor cells in a FAP dependent manner, with low binding to normal cells.